We describe a one-step method for creating thousands of isolated pico- to microliter-sized droplets with defined geometry and volume. Arrays of droplets are instantly formed as liquid moves along a superhydrophilic-superhydrophobic patterned surface. Bioactive molecules, nonadherent cells, or microorganisms can be trapped in the fully isolated microdroplets for high-throughput screening, or in hydrogel micropads for screening in 3D microenvironments.